The dominant Australian community-acquired methicillin-resistant Staphylococcus aureus clone ST93-IV [2B] is highly virulent and genetically distinct.
The Dominant Australian Community-Acquired Methicillin-Resistant Staphylococcus aureus Clone ST93IV [2B] Is Highly Virulent and Genetically Distinct Kyra Y. L. Chua1,2,4, Torsten Seemann3, Paul F. Harrison3, Shaun Monagle6, Tony M. Korman7, Paul D. R. Johnson4, Geoffrey W. Coombs8, Brian O. Howden9, John K. Davies1, Benjamin P. Howden1,2,4,5*., Timothy P. Stinear1,2*. 1 Department of Microbiology, Monash University, Clayton, Australia, 2 Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia, 3 Victorian Bioinformatics Consortium, Monash University, Clayton, Australia, 4 Department of Infectious Diseases, Austin Centre for Infection Research, Austin Health, Heidelberg, Australia, 5 Department of Microbiology, Austin Health, Heidelberg, Australia, 6 Department of Anatomical Pathology, Eastern Health, Box Hill, Australia, 7 Department of Infectious Diseases, Monash Medical Centre, Clayton, Australia, 8 Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine WA, Royal Perth Hospital, Perth, Australia, 9 Microsurgical Consultants, Blackburn, Australia Abstract Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding a-hemolysin, Panton-Valentine leukocidin (PVL) and a-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone’s enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a highly conserved accessory genome from a common source. Citation: Chua KYL, Seemann T, Harrison PF, Monagle S, Korman TM, et al. (2011) The Dominant Australian Community-Acquired Methicillin-Resistant Staphylococcus aureus Clone ST93-IV [2B] Is Highly Virulent and Genetically Distinct. PLoS ONE 6(10): e25887. doi:10.1371/journal.pone.0025887 Editor: Malcolm James Horsburgh, University of Liverpool, United Kingdom Received June 28, 2011; Accepted September 12, 2011; Published October 3, 2011 Copyright: ß 2011 Chua et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the National Health and Medical Research Council of Australia Project Grant 1008656, the CASS Foundation and the Austin Hospital Medical Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: email@example.com (TPS); Benjamin.Howden@austin.org.au (BPH) . These authors contributed equally to this work. genome such as Panton-Valentine leukocidin (PVL), a-hemolysin (Hla) and a-type phenol soluble modulins (PSMs) [3,4,5,6,7, 8,9,10]. In contrast to healthcare-associated MRSA where a limited number of clones are prevalent, CA-MRSA strains are polyclonal with an epidemiological association between clone type and geographic origin [1,11]. Furthermore, minor genetic differences between CA-MRSA strains, even to a single nucleotide, may result in significant differences in the observed virulence of a strain . Hence, while the genomic mediators of virulence have been well defined in USA300, these may not be applicable to other CAMRSA clones. Therefore, a detailed understanding of the virulence phenotype and virulence determinants in other global Introduction Infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in patients with no contact with the hospital environment are now well described worldwide . In some regions such as North America, the CA-MRSA phenomenon has become particularly well defined. Here, the epidemic CA-MRSA strain USA300 is the most common cause of skin infections in patients presenting to the emergency department . Much of the work performed thus far to understand the CA-MRSA phenomenon has focused on USA300 and these studies have demonstrated the importance of virulence determinants in both the staphylococcal accessory and core PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25887 Functional and Genomic Analysis of CA-MRSA ST93 killing assay and the mouse skin infection assay, S. aureus were harvested at the stationary phase of growth after 18 hours incubation (OD600 approx. 2.0), washed, diluted and resuspended in PBS. The bacterial inoculum (CFU) and viable counts were determined by plating onto BHI agar and colony enumeration. clones of CA-MRSA has the potential to provide new and important insights into staphylococcal pathogenesis. In Australia, S. aureus ST93-IV [2B] has recently emerged to become the dominant CA-MRSA clone. First described in the early 2000s, it is colloquially known as the ‘‘Queensland’’ clone . However, since this initial description, ST93-IV [2B] has been reported across Australia and is responsible for the increasing prevalence of all CA-MRSA nationwide . ST93 is associated with skin infection and severe invasive infection including necrotizing pneumonia, deep-seated abscesses, and septicemia [14,15]. This PVL positive clone is a singleton by MLST eBURST analysis and is therefore distinct to other S. aureus clones, including previously sequenced strains . While predominantly an Australian strain, it has recently also been reported in the United Kingdom, with many cases having epidemiological links to Australia [16,17]. Other less frequent clones of CA-MRSA in Australia include PVL negative ST1-IV [2B] (WA-1) and PVL positive ST30-IV [2B] (SouthWest Pacific, SWP) . The dominant health-care associated clone of MRSA in Australia is multi-resistant ST239-III [3A] . To investigate the apparent enhanced clinical virulence of ST93-IV [2B], we examined a recent clinical isolate (JKD6159) that caused an outbreak of both severe and minor staphylococcal infections within a household, representative of the typical spectrum of disease associated with this clone. We compared the virulence of JKD6159 to three other well-characterized and representative Australian MRSA strains and the epidemic North American strain, USA300 and found that JKD6159 was the most virulent clone in two in vivo models. We next sequenced, fully assembled and annotated the genome of S. aureus ST93-IV [2B] (JKD6159) and then sequenced three non-ST93 Australian CA-MRSA clones. We employed comparative genomics against these strains, the 19 published, complete S. aureus genomes and 59 unassembled, publically available S. aureus genomes to try and identify genetic factors that might explain the recent emergence, and increased virulence of ST93-IV [2B]. DNA Manipulation and Molecular Typing DNA was extracted using the GenElute kit according to the manufacturer’s instructions (Sigma-Aldrich). Detection of lukSF-PV was performed as described by Lina et al. . MLST was performed as described by Enright et al. , and Pulsed Field Gel Electrophoresis (PFGE) performed as previously described . SCCmec typing was performed as previously described . SCCmec nomenclature was used as proposed by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) . A Roman numeral indicates the SCCmec type with a lowercase letter indicating the subtype. The ccr complex and the mec complex are indicated by an Arabic numeral and an uppercase letter respectively. Wax Moth (Galleria mellonella) virulence assay Final stage instar G. mellonella larvae were infected with S. aureus and time to death measured as described previously . Approximately 106 CFU of S. aureus suspended in 10 mL of PBS was injected into the first left proleg of each larva. Each experimental group consisted of at least 16 larvae. All experiments included biological triplicates. The larvae were incubated at 37uC and assessed daily for six days. Larvae were considered dead when there was no movement in response to stimuli. The control groups were PBS injected and non-injected larvae. Mouse skin infection assay Mice were housed in individually ventilated cages and received food and water ad libitum. Six-week-old female BALB/c mice were anesthetized with intraperitoneal ketamine/xylazine, and inoculated in the right flank by intradermal injection, with 108 CFU of S. aureus (in 50 mL of PBS suspension). This dose of S. aureus was determined in preliminary experiments as the lower limit of inoculum, which produced consistent dermonecrosis for all strains of S. aureus tested. Mice were assessed and weighed daily for five days. On the 5th day, the mice were culled. The infected area of skin and muscle was harvested and these tissues were homogenized and CFU measured by plating onto BHI and colony enumeration. Skin lesion area was quantitated by obtaining a digital image of the lesion and processed using ImageJ . For each S. aureus strain, at least 15 mice were assessed – the tissues for four of these mice were used for histological analysis and data for these mice were not available for CFU enumeration. Materials and Methods Bacterial strains and culture The bacterial strains used in this study are described in Table 1. For all experiments, bacteria were grown in brain heart infusion broth (BHI, Oxoid). For growth kinetic experiments, overnight cultures of S. aureus were diluted 1:100 and incubated at 37uC with shaking (180 rpm). Optical density at 600 nm (OD600) and viable counts were performed hourly until stationary phase was achieved and at 24 hours after commencement of the experiment. These experiments were performed in triplicate. For the Galleria mellonella Table 1. Bacterial strains used in this study. S. aureus strain Type Date of Isolate Recovery Place of Isolate Recovery Relevant Characteristics lukSF-PV Reference JKD6159 ST93-IV [2B] 2004 Melbourne, Australia Dominant Australian CA-MRSA clone + This work JKD6272 ST1-IV [2B] 2002 Melbourne, Australia Australian CA-MRSA clone 2 This work JKD6260 ST1-IV [2B] 2008 Port Hedland, Australia Australian CA-MRSA clone + This work JKD6177 ST30-IV [2B] 2003 Melbourne, Australia Australian CA-MRSA clone + This work FPR3757 USA300 ST8-IV [2B] NA San Francisco, USA Dominant North American CA-MRSA clone +  JKD6009 ST239-III [3A] 2002 New Zealand Dominant Australian hospital-associated MRSA clone, AUS2/3  2 NA: not available. doi:10.1371/journal.pone.0025887.t001 PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e25887 Functional and Genomic Analysis of CA-MRSA ST93 Ethics Statement Table 2. Staphylococcus aureus completed genomes used in this study. All studies were reviewed and approved by the Animal Ethics Committee at the University of Melbourne approval number 0911248.2. Whole Genome sequencing of Australian CA-MRSA strains JKD6159 (ST93-IV [2B]), JKD6272 (ST1-IV [2B]), JKD6260 (ST1-IV [2B]) and JKD6177 (ST30-IV [2B]) Genome We have previously described the sequencing, complete assembly and annotation of the genome of JKD6159, however the comparative genomics have not previously been reported . Here, we also sequenced and partially assembled the genomes of three Australian CA-MRSA strains, using Illumina GAIIx 36 bp paired-end chemistry, yielding an average of 555 Mbp of reads per strain (,2006 coverage). These genomes were de novo assembled to draft form using Velvet 1.0 . The reads from whole genome sequencing of these strains were also included in defining the core and accessory genome of JKD6159. The sequences are accessible from the NCBI Sequence Read Archive under accession SRA026511.1. Analysis of the JKD6159 genome sequence A read mapping approach was developed to define a S. aureus core genome and the accessory genome for a given strain. Sequence reads from each S. aureus strain were aligned separately to the completed JKD6159 reference genome using SHRiMP 2.0 . Publically available completed genomes (Table 2) were shredded into 75 bp reads at 256 coverage to produce a set of synthetic sequence reads. Those positions in JKD6159 that were covered by at least one read from each and every genome defined a S. aureus core genome. Reads from JKD6159 that did not map to any S. aureus genome (Table 2) were used to define JKD6159 regions of difference. Reads from any strain that did not map to the core genome represented that strain’s accessory genome. SNPs were identified using Nesoni v0.35, which used the sequence reads for each genome aligned to the above-defined core genome to construct a tally of putative differences at each nucleotide position, including substitutions, insertions, and deletions (www.bioinformatics.net.au). This tally was then employed in a simple Bayesian model to decide whether a base (or deletion) could be called for the position, and if so, whether it differed from the reference sequence. A similar procedure was used to determine the presence or absence of insertions between positions in the reference. NCBI BLASTn+, Artemis and BRIG were also used for comparisons of JKD6159 with selected S. aureus genomes  (http://sourceforge. net/projects/brig/). SNPs were identified in the accessory genome elements pMW2, wSA2, and SCCmecIV for strains carrying them by read mapping using Nesoni v0.35 against reference sequences of each element taken from S. aureus MW2. Phylogenetic analyses were performed using a distance method, based on pairwise nucleotide sequence alignments for the S. aureus core genome among all strains or selected accessory elements among some strains. Split decomposition analysis was employed using uncorrected p distances with bootstrapping as implemented in SplitsTree4 . Reference 1 NCTC8325 8 (8)  2 N315 5 (5)  3 Mu50 5 (5)  4 Mu3 5 (5)  5 MW2 1 (1)  6 USA300 FPR3757 8 (8)  7 USA300 TCH1516 8 (8)  8 MRSA252  9 MSSA476 36 (30) 1 (1)  10 RF122 151  11 Newman 8 (8)  12 COL 250 (8)  13 JH1 105 (5)  14 JH9 105 (5)  15 ED98 5 (5)  16 TW20 239 (8)  17 04-02981 225 (5)  18 S0385 398  19 JKD6008 239 (8)  doi:10.1371/journal.pone.0025887.t002 Results Clinical Details Staphylococcus aureus JKD6159 was isolated from the blood of a young male intravenous drug user with no prior health care exposure, who presented with a severe sepsis syndrome in 2004. He had cavitating pulmonary lesions, polyarticular septic arthritis and multiple deep-seated muscle abscesses. The isolate was resistant to methicillin, but susceptible to erythromycin, clindamycin, tetracycline, ciprofloxacin, rifampicin, fusidic acid and vancomycin. The patient required extensive surgical debridement and prolonged antimicrobial therapy with vancomycin and subsequently, linezolid, but eventually made a full recovery. In addition, the patient reported multiple household family members with recurrent skin infections. The SmaI PFGE pattern was identical between JKD6159 and the S. aureus isolates from three other family members, indicating an intra-familial outbreak with the same clone (Figure 1). Further typing demonstrated that JKD6159 was ST93-IV [2B]. This isolate was representative of the Australian CA-MRSA Queensland clone. S. aureus strain JKD6272 was isolated from the blood of a woman with pneumonia and septic shock who had no prior hospitalizations. The isolate was resistant to methicillin and fusidic acid but susceptible to erythromycin, clindamycin, tetracycline, ciprofloxacin, rifampicin, trimethoprim and vancomycin. Further typing demonstrated that JKD6272 was ST1-IV [2B], and PVL negative. This isolate was representative of the Australian CAMRSA clone WA-1. S. aureus strain JKD6260 was isolated from a wound swab from a patient with a skin infection. The isolate was resistant to methicillin, fusidic acid and ciprofloxacin, but susceptible to erythromycin, clindamycin, tetracycline, rifampicin, mupirocin, Statistical analysis Kaplan Meier plots of G. mellonella killing results were analysed using the log rank test. Percentage mouse weight change at day 5, viable counts of S. aureus in mouse tissues and skin lesion area of foram contidos fisicamente em decúbito lateral direito, e as agulhas posicionadas sobre os acupontos (ACs) coração 7 (C-7 – Shenmen), localizado entre o processo estiloide lateral e o osso cárpico acessório, exatamente na dobra da articulação úmero-rádio-ulnar, e pericárdio 6 (PC-6 – Neiguan), localizado entre os tendões do músculo flexor carporradial e do músculo flexor digital superficial (Fig. 1A, 1B e 1C), conforme descrito por Draehmpaehl e Zohmann (1997). Já para a segunda seção, os animais foram novamente posicionados em decúbito lateral direito, e as agulhas inseridas em acupontos falsos (AF), localizados no oitavo e no nono espaço intercostal esquerdo (Figura 1D), onde não há interferência de nenhum outro meridiano, conforme descrito por (Draehmpaehl e Zohmann, 1994). Independentemente da seção, não foi realizada estimulação manual das agulhas, e os acupontos permaneceram estimulados por um período de 30 minutos, respeitando-se o período mínimo de 10 a 20 minutos sugerido por Scott (2001) para que o acuponto possa promover um efeito observável. Figura 1. Localização dos acupontos C-7 (seta em A) e PC-6 (seta em B) no cão (Adaptado de Draehmpaehl e Zohmann, 1997). Em C, acupuntura em cão do experimento acuponto C-7 (seta azul) e acuponto PC-6 (seta laranja). Em D, acupuntura em cão do experimento em acuponto falso. Arquivo pessoal, 2014. Previamente à aplicação das agulhas nos ACs, foi realizado um registro eletrocardiográfico (ECG) com dois minutos de duração e, após a inserção das agulhas nos ACs, o registro eletrocardiográfico se manteve durante os 30 minutos de acupuntura. Para a realização do eletrocardiograma, foi utilizado um aparelho de eletrocardiografia computadorizada (ECGPC – TEB®), respeitando-se o padrão de fixação de eletrodos descrito por Tilley (1992), com registro das derivações bipolares e unipolares aumentadas e calibração de 25mm/s e 1mV (N). Os dados do ECG foram interpretados de forma comparativa pré e pós-estimulação dos acupontos, sendo os dados pós-estimulação coletados e interpretados em intervalos de cinco minutos até o final da gravação (30 minutos). Arq. Bras. Med. Vet. Zootec., v.68, n.1, p.252-256, 2016 253 Silva et al. Para a análise da VFC, foi utilizada a fórmula disponível em Carareto et al. (2007). A normalidade dos dados obtidos foi verificada pelo teste de Shapiro-Wilk (P>0,05), seguida pelo teste ANOVA com pós-teste de Bonferroni para as variáveis paramétricas e pelo teste de Kruskal-Wallis com pós-teste de Dunn (P<0,05) para as variáveis não paramétricas. Foi realizada análise descritiva para as variáveis qualitativas. No presente estudo, 90% dos animais apresentaram arritmia sinusal respiratória, demonstrando maior influência do sistema nervoso simpático, em todos os momentos do grupo ACs, enquanto no grupo AF esse ritmo cardíaco esteve presente em 100% dos animais, em todos os momentos. O ritmo sinusal foi encontrado apenas em 10% dos animais do grupo ACs, sugestivamente por se tratar da primeira exposição dos animais ao experimento, resultando em maior influência parassimpática sobre o sistema cardiovascular. Conforme demonstrado na Tab. 1, houve pequena e irrelevante diferença quando comparada a FC de todos os momentos do grupo ACs com os momentos do grupo AF, o que evidencia, portanto, a ineficácia da estimulação dos acupontos sobre esse parâmetro. Tabela 1. Frequência cardíaca (FC) e variabilidade da frequência cardíaca (VFC) obtidas com a realização dos acupontos verdadeiros (AV) C-7 e PC-6 e do acuponto falso (AF) em diferentes momentos do ECG, expressos em mediana (percentil 25% - percentil 75%) para a FC e média±desvio-padrão para a VFC Momentos FC (bpm) VFC AV AF AV AF 0 minuto 107 (92-130)a 112 (93-129)a 12,79±0,46ab 12,71±0,47b 5 minutos 90 (79-121)a 93 (82-115)a 13,08± 0,48ab 13,09± 0,49ab 10 minutos 94 (79-118)a 88 (70-123)a 13,01± 0,48ab 13,12± 0,53ab 15 minutos 96 (76-120)a 99 (81-115)a 13,04± 0,52ab 13,17± 0,51a 20 minutos 95 (79-120)a 95 (81-112)a 13,13± 0,51ab 13,07± 0,54ab 25 minutos 104 (83-115)a 89 (76-108)a 13,15± 0,45ab 13,17± 0,53a 30 minutos 87 (82-107)a 95 (81-117)a 13,18± 0,46ab 13,11± 0,56ab P = 0,0890 P = 0,0051 Para a FC, valores seguidos por letras distintas diferem entre si (P<0,05) ao teste de Dunn. Para a VFC, valores seguidos por letras distintas diferem entre si (P<0,05) ao teste de Bonferroni. Não foi observada diferença na VFC (Tab. 1) entre os grupos do ACs e AF, exceto quando comparado apenas o momento basal com os momentos 15 e 25 minutos do AF, contudo sem relevância clínica para o estudo. Sabidamente, VFC constitui uma medida simples e não invasiva que avalia os efeitos do sistema nervoso autônomo sobre o coração. Variações nessa variabilidade são normais e esperadas, indicando habilidade do coração em responder a múltiplos estímulos fisiológicos e ambientais. Em síntese, uma alta VFC representa boa adaptação, caracterizando um indivíduo saudável, com mecanismos autonômicos eficientes, enquanto a baixa VFC representa adaptação anormal e insuficiente do SNA (Carareto et al., 2007). Dentre as opções terapêuticas para o tratamento de anormalidades dos mecanismos autonômicos, a acupuntura vem se destacando em vários estudos em animais, entretanto em nenhum desses estudos se objetivou a avaliação do efeito da acupuntura em indivíduos saudáveis, conforme ocorreu no presente estudo, fator limitante à discussão mais aprofundada. Observam-se, na literatura, diversos estudos em que foram observados os efeitos da acupuntura sobre pacientes com diferentes distúrbios cardiovasculares. Syuu et al. (2001) relataram efeitos benéficos da estimulação do acuponto PC-6 em cães anestesiados e sob toracotomia. Nesse estudo, os autores descrevem reduções na pressão arterial média, volume diastólico final, frequência cardíaca, débito cardíaco e pressão sistólica final mediante estimulação do acuponto. O efeito cardiovascular da acupuntura sobre o acuponto PC-6 em cães também foi observado por Jingbi (2004) em modelo de experimental de infarto agudo do miocárdio. Nesse estudo, foi observado que a acupuntura proporcionou redução do segmento ST do eletrocardiograma, além de reduzir as áreas de infarto induzidas pela ligadura coronariana. Entretanto, esses dois 254 Arq. Bras. Med. Vet. Zootec., v.68, n.1, p.252-256, 2016 Efeitos da acupuntura estudos diferem do presente pelo fato de as agulhas terem sido estimuladas eletricamente (eletroacupuntura), enquanto no presente estudo estas permaneceram em repouso. Cárdenas (2006) observou que a estimulação dos mesmos acupontos empregados no presente estudo em cavalos sob anestesia geral demonstrou efeitos positivos sobre a taquicardia ventricular induzida por infusão contínua de dopamina, nos quais a estimulação dos ACs C-7 e PC-6 promoveu diminuição do tempo da taquicardia ventricular, demonstrando, assim, a eficácia da estimulação desses acupontos em animais. Um estudo controlado em ratos demonstrou que a estimulação dos acupontos PC-5 e PC-6 reduziu significativamente a incidência de taquiarritmias induzidas experimentalmente pós-oclusão e reperfusão da artéria coronária esquerda (Lujan et al., 2007). Em humanos, a revisão de oito estudos utilizando acupuntura para o tratamento de arritmias cardíacas encontrou cerca de 87 a 100% que foram convertidos ao ritmo sinusal após a estimulação dos acupontos (Xie e Wedemeyer, 2012). estudo não se valeu da estimulação elétrica sobre os acupontos falsos ou verdadeiros (C-7 Shenmen e PC-6 Neiguan). Limitações a este estudo compreendem o fato de que a escolha dos acupontos explorados baseouse nas indicações terapêuticas descritas por Draehmpaehl e Zohmann (1994), em que o acuponto C-7 é indicado em casos de alterações funcionais do coração, e o acuponto PC-6 quando há dores miocárdicas. Ainda segundo os mesmos autores, essa forma de terapia alternativa objetiva normalizar funções orgânicas alteradas e, como no presente estudo foram utilizados animais hígidos, a acupuntura pode não ter promovido influência sobre a VFC nos cães que participaram do experimento. Limita-se, ainda, este estudo quando da observação das recomendações descritas por Scott (2001), quando ele destaca a necessidade de um ou dois tratamentos semanais durante quatro a seis semanas consecutivas, tempo em que se espera que possa haver a resposta desejada à terapia, fato não realizado no presente estudo. Finalmente, Kimura e Hara (2008) desenvolveram um estudo em que foram estudados os efeitos da eletroacupuntura sobre o ritmo do sistema nervoso autônomo em cães. 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The role of vocabulary size in assessing second language proficiency. MA Thesis, Brigham Young University, 2004. 121 APPENDIX 122 APPENDIX 1 – THE ACCEPTABILITY JUDGMENT TASK FOR EXPERIMENT 1 The man laughed the children during the party. The scientist appeared her paper after years. The director spoke the actor about the play. The woman played the children until bed time. The farmer fell the apple from the tree. The teenager arrived his friend for his appointment. The staff flowered the plants before spring. The president resigned the minister after the incident. The girl give the cats milk twice a day. Does the professor talk often about his research? Where are the articles that contains the information? Don have always written nice poems. Who did Jane call her friend after she saw? What did Steven read the book that Helen talked about? Moses imagined to whom what he said. Which case did the detective say was worried? The instructor ran the boys around the park. The lady walked her child down the street. The man swam his dog to his boat. The rider jumped his horse over the fence. The scientist flew her balloon above the clouds. The captain marched his troop into the town. The child floated her toys on the pond. The woman danced the man across the room. The coach told the players to jog around the field. The officer warned the tourists not to drive in the park. The instructor asked the girls to skate across the lake. The veterans ordered the recruits to leap over the hole. The guards forced the prisoners to march through town. The teacher told the students to wait for the bell. The pilots made the planes loop in the sky. The policeman told the driver to fix his brake lights. The girls melted the cheese in the bowl. The fighter broke the chair in the corner. The servant dried the sheets in the garden. The woman baked her bread at her place. The cook froze his dinner at the restaurant. The general burned the bridges to the village. The students cooled the cakes on the sink. The man warmed his soup on the stove. The ball rolled down the street for a few minutes. The horse leaped over the fence without warning. The athletes paraded along the avenue after the victory. The kids played in the park all morning long. The boys walked by the stadium at night. The teacher graded the students’ essays at home. The president spoke about the crisis to the press. The workers returned to the factory after two days. The actress answered the questions of the interviewer. The doctor neglected the patient’s complaints about his headache. The students read the novels over the semester. The clerks cleaned the office after work. The driver took the visitors to the wrong place. The nurse fed the baby every three hours. The hunter killed the deer with a gun. The criminals burned the papers from the safe. 123 APPENDIX 2 – THE ENGLISH VERSION OF THE VOCABULARY LEVEL TEST (VLT) 124 125 126 APPENDIX 3– THE SPANISH VERSION OF THE VOCABULARY LEVEL TEST (VLT) 127 128 APPENDIX 4– THE OXFORD PLACEMENT TEST (OPT) 129 130 131 132 133 134 135 136 137 APPENDIX 5– THE SPANISH PLACEMENT (SPT) 138 139 140 141 142 143 APPENDIX 6 - THE BILINGUAL HISTORY QUESTIONNAIRE (BHQ) 144 145